1. Field of the Invention
The present invention relates to biotechnology field, more specifically to genetic engineering in plants. The invention provides useful DNA sequences and constructions to regulate recombinant gene expression in plants. More specifically, the invention provides new regulatory sequences derived from Arabidopsis thaliana COX5c-1, COX5c-2 and COX5c-3 genes.
2. Description of the Prior Art
Genetic engineering in plant biotechnology made an amazing advance in the fields of research and production of new products.
Research in genetic engineering requires access to a wide variety of sequences that are useful for the regulation of transgenes. There are two examples of such elements: introns and promoters.
Selection of promoters controlling or directing transcription levels of one or more genes is one of the greatest challenges that molecular biology must achieve for the success of plant biotechnology.
Therefore, many efforts have been made in the last two decades to find promoters and other sequences capable of guaranteeing the expression according to the needs of each transgene. Several promoters of different species have been studied for production of transgenic plants; including vegetal, viral, from the Ti and Ri plasmids of Agrobacterium tumefaciens, as well as natural exons and introns, namely the first intron of maize alcohol dehydrogenase-1 (Callis et al., 1987) or the first exon/intron of rice actin-1 gene (McElroy et al., 1991).
Gene transcription is regulated by a promoter region (cis element) and multiple regulatory proteins (trans elements). A genetic engineering project requires the simultaneous use of several promoters. A first promoter can be used upstream of the gene of interest, while a second promoter can be used to express a selection marker.
Eukaryotic genes are usually interrupted by non-coding sequences, the introns. Eukaryotic genes are first transcribed as pre-RNA, which contain introns within their sequences. Introns are then removed during the splicing, the final product, the mRNA, can be translated to a protein.
It has been shown that introns are involved in the regulation of gene expression in plants. The first intron of maize enzyme alcohol dehydrogenase-1 (Adh-1) gene is capable of increasing transcription in anaerobiosis (Callis et al., 1987). Although in a smaller degree, the intron also stimulates transcription in aerobic conditions. It has been proposed that the enhancement of gene expression is due to pre-RNA stabilization produced by the introns. Remarked increases have also been reported in the expression of gene CAT (from 12 to 20 times) when the first intron of the maize dehidrogenase alcohol (Mascarenhas et al, 1990). It has also been reported that these effects are produced at the transcription level.
Naturally occurring introns with their adjacent sequences have been employed to increase transcription, especially when intron is located near the 5′ end of the gene. It has also been reported that potentiation of intron-mediated mRNA accumulation (IME) depends on intron origin, exonic regions flanking said intron and cell type. Molecular mechanisms underlying IME have not been completely elucidated (Simpson et al., 1996; Lorkovic et al., 2000).
Enhancement of expression by introns has been reported for several genes from maize and other monocots (see WO98/5921, and also see Callis et al., 1987; McElroy et al., 1990; Christensen et al., 1992; Xu et al., 1994; Jeon et al., 2000; Morello et al., 2002) and dicot plants (Norris et al., 1993; Gidekel et al., 1996; Rose and Last, 1997; Plesse et al., 2001; Mun et al., 2002). Introns influencing expression are more frequently located near the translation start site within non-coding regions, as is the case for COX5c genes. The exact role of introns in promoting an increase in expression levels is not clear. Some introns seem to contain transcriptionally active regulatory elements (Gidekel et al., 1996), while others seem to act post-transcriptionally (Rose and Last, 1997), suggesting the existence of different mechanisms of action. It has recently been proposed that many introns would act by increasing the processivity of the transcription machinery (Rose, 2004). Besides the quantitative enhancement of expression, some introns direct tissue-specific patterns of expression (Bolle et al., 1996; Jeon et al., 2000). In some cases, like those of the Petunia actin-depolymerizing factor (Mun et al., 2002), the rice α-tubulin OstubA1 (Jeon et al., 2000) and the Arabidopsis polyubiquitin Ubi1 y Ubi4 genes (Plesse et al., 2001), expression is specifically observed in vascular tissues and/or metabolically active dividing cells. These expression patterns are similar to those observed here for the COX5c genes, probably indicating that these introns operate with similar mechanisms or respond to similar factors.
Although the involvement of introns in translation seems an unexpected consequence, similar observations have been made in animal and plant systems (Le Hir et al., 2003; Rose, 2004). It has been proposed that increment in translational efficiency by introns is related to the location of proteins near the exon-exon junctions during splicing that subsequently increase the interaction of ribosomes with the mRNA (Wiegand et al., 2003; Nott et al., 2004).
Cytochrome c oxidase (COX) is a multimeric complex composed of several different subunits, two or three of them encoded by the mitochondrial genome and the rest encoded in the nucleus (Grossman et al., 1997; Jänsch et al., 1996).
Three different nuclear-encoded subunits, COX5b, COX6a, and COX6b, have been identified in plants through sequence comparisons with yeast and animal counterparts (Kadowaki et al., 1996; Ohtsu et al., 2001; Curi et al., 2003).
A fourth subunit, COX5c, is the smallest COX plant subunit and has been discovered by protein purification studies (Nakagawa et al., 1987, 1990). Recent studies using 2D gel electrophoresis combined with mass spectrometry indicated the presence of additional plant-specific subunits (Millar et al., 2004).
COX5c is a polypeptide of about 63 amino acids with sequence similarity to yeast COX VIIa and mammalian COX VIII (Nakagawa et al., 1990). COX5c cDNAs have been isolated from sweet potato, rice, and sunflower (Nakagawa et al., 1990; Hamanaka et al., 1999; Curi et al., 2002), and ESTs from several species are available. The first COX5c gene was also isolated from sweet potato (Nakagawa et al., 1993), and related sequences could be detected in the totally or partially sequenced genomes from Arabidopsis, rice, and Lotus corniculatus. Expression studies in rice and sunflower indicated that COX5c genes are expressed at different levels throughout the plant (Hamanaka et al., 1999; Curi et al., 2002). However, no detailed analysis on tissue specificity of expression or on the gene sequences involved in directing this expression have been performed by any plant COX5c gene.
It is generally assumed that the expression of components of the plant mitochondrial respiratory chain must somehow be co-ordinated. It is now well established that most mitochondrial components show enhanced expression in flowers (Huang et al., 1994; Landschütze et al., 1995; Felitti et al., 1997; Heiser et al., 1997; Zabaleta et al., 1998). Expression in flowers is mainly localized in anthers as indicated by in situ hybridization experiments (Smart et al., 1994; Ribichich et al., 2001; Elorza et al., 2004). Expression studies in Arabidopsis thaliana have shown similar responses for the nuclear genes encoding cytochrome c and COX subunits 5b, 6a, and 6b (Welchen et al., 2002; Curi et al., 2003). Notably, a different behaviour has been observed for genes encoding COX subunit 5c (COX5c), at least in sunflower (Curi et al., 2002). However, no functional studies have been performed on the cis-acting sequences required for the expression of COX5c genes from any species.